Sporadic colorectal cancer (CRC) remains a global health burden as one of the leading causes of cancer and cancer-related deaths, and, strikingly, is also emerging as an acute health crisis in younger patients in the US under the age of 50. These data suggest that novel efforts to identify contributors to the changing landscape of CRC are urgently needed. In this K99/R00 proposal, we seek to dissect the assembly and tumorigenic mechanisms behind invasive, polymicrobial colonic biofilms, which were previously established in the Sears laboratory to be a nearly universal feature of CRC tumors proximal to the hepatic flexure. Biofilms were also identified on a subset (10-15%) of healthy patients without CRC, in whom biofilms were associated with early, procarcinogenic changes, positing a role for bacterial biofilms in CRC development. These biofilms were also associated with elevations in polyamine metabolites in mass spectrometry-based analyses. In our new preliminary data, we demonstrate that slurries made from human biofilms can re-assemble into biofilms in the distal mouse colon and are directly tumorigenic in germ-free mice by 10-15 weeks. These biofilms are also shown to induce a strong Interleukin-17 (IL-17) response in the mice by one week, a cytokine signature associated with a worse prognosis in human CRC. Our specific aims seek to build on these preliminary studies. Specific Aim (SA) 1 will identify the organisms present in the mouse distal colon mucosa of germ-free ApcMin/+ mice at various time points post-inoculation with biofilm-positive or biofilm- negative human mucosal slurries, using a combination of fluorescence in situ hybridization and culturing methods complexed with rapid identification using matrix-assisted laser desorption ionization (MALDI) time-of- flight (TOF) mass spectrometry. Bacterial isolates and mouse mucosal slurries will be re-inoculated into additional mice to confirm the biofilm-forming and tumorigenic organisms. SA2 will test the hypothesis that IL- 17 is a critical mediator of biofilm formation and biofilm-mediated tumorigenesis. Tissues from SA1 will be used to determine the peak in IL-17a mRNA induction by qRT-PCR in response to the human mucosal biofilm slurries, and additional mice will be repeated at these time points in order to examine the induction of T helper subsets and critical cytokines by flow cytometry. Therapeutic modulation with an IL-17 antibody or knockout mice will be used to confirm the requirement of IL-17 in biofilm formation and biofilm-mediated tumorigenesis. SA3 will utilize advanced mass spectrometry methods to determine metabolites associated with biofilms in both patient and mouse tissues and the localization of these metabolites in select samples. Overall, the experiments in this proposal will provide valuable information regarding the host and microbial requirements for biofilm formation and biofilm-mediated tumorigenesis that will inform the field as to potential therapeutic targets or biomarkers for biofilms. The studies and training described in this proposal will also provide Dr. Drewes with critical datasets, clinically relevant bacterial isolates, and metabolites of interest for future R01 applications.